Myosins are the molecular motors that power muscle. They are also responsible for many events inside cells where they are involved in cell division (cytokinesis), targeting cargos to different regions of the cell (vesicle trafficking) and the development and maintenance of cellular architecture. Defects in myosins can lead to life threatening disease. Over the last decade, a synthesis of structural, biochemical and biophysical data has resulted in a general model for how these molecular motors work. Many of the experiments proposed in this application are designed to test fundamental aspects of the model that have been called into question by some recent publications. Cryo-electron microscopy and image analysis will be used to examine recombinant myosin VI motors to determine which regions of the molecule determine directionality, processivity and step size. Additional experiments will reveal the organization, structure and regulatory role of the myosin VI light chain binding domain, the structure and actions of myosin IX, and the ADP response of a myosin I. Collaborators will provide native and recombinant proteins. Planned technical developments include using a single particle image processing approach to increase the resolution attainable in the 3D maps.